Molecular Probes, Inc., has developed a fluorescence-based, Multiplexed Proteomics (MP) platform that allows for the simultaneous identification and characterization of glycosylation, phosphorylation and total protein in a single 2-D gel. With subpicomolar sensitivity, the system provides for the rapid quantification of differentially regulated and/or post-translationally modified proteins, with subsequent mass spectrometry based identification. The central objective of this proposal is to utilize this technology to discover the important protein targets which confer pregnancy-induced protection against mammary carcinogenesis. Full-term pregnancy early in life is the most effective natural protection against breast cancer in women, and in experimental models, rats are similarly protected. It is proposed that these pregnancy-specific changes in susceptibility to mammary tumorigenesis are ultimately due to changes in the mammary epithelial proteome and include altered post-translational regulation and expression of key signaling proteins involved in maintaining cellular homeostasis and protection from cellular transformation. There have been no published comprehensive proteomic studies aimed at determining the molecular origin of pregnancy-induced mammary carcinogenesis protection. Our MP platform is uniquely capable of rapid detection and quantitation of changes in the posttranslational modification state of proteins, an event which we believe is crucially linked to the understanding of this effect. Phase I studies will characterize protein-based changes that take place in the virgin rat mammary epithelial cell proteome in response to full-term pregnancy or treatment with pregnancy levels of ovarian hormones. A fully integrated approach to sample preparation, 2-D gel/mass spectrometry techniques, and data analyses is demonstrated. A minimum of three protein targets linked to pregnancy-induced protection will be identified. Using a combination of laser-tissue microdissection and antibody-based blotting techniques, these initial protein targets will be independently validated. Building upon this validated proteomics-based discovery platform, Phase II of this program will expand its efforts to characterize and classify the important signaling cascades and protein targets linked to the pregnancy-induced cancer protection phenotype. These protein targets will be ideal candidates for the rational design of diagnostic indicators and/or therapeutic intervention.